SSC6b

The association analysis revealed 22 significant SNPs (P < 0.05) in the SSC6b region (Table 8, Appendix V); 8 of these are novel SNPs detected by resequencing, and 14 are pre-existing markers from Illumina’s PorcineSNP60 BeadChip. Twelve SNPs are located in gene regions, affecting a total of 6 genes. Two genes are so far uncharacterized; the remaining genes are nuclear factor I/A (NFIA), chromosome 6 open reading frame, human C1orf87 (C6H1orf87) and two FGGY carbohydrate kinase domain containing genes (FGGY). All SNPs have according to SnpEff (v2.1a) a “modifier” effect, which is the lowest level of impact. None of the novel SNPs on the priority list (2 missense SNPs and 2 synonymous SNPs) were significantly associated with inguinal hernia.

Table 8: Association analysis results of the significant SNPs in region SSC6b together with genes affected by the SNPs. SNPs within a gene region are marked with grey.

SNP name Chr. position p-value Region/Genes Gene description MARC0037985 138852864 0.030

NFIA

(138764565-139164050) Nuclear factor I/A 6_138924570 138924570 0.033

6_139077739 139077739 0.045

6_139626802 139626802 0.014 Intergenic region 6_139664663 139664663 0.012 ALGA0115609 140397044 0.031 FGGY

(140364020-140400250) 6_143795121 143795121 0.050 Intergenic region

ASGA0095712 143814685 0.050 Intergenic region 6_143889528 143889528 0.033 Intergenic region MARC0016562 143952619 0.029

LOC102158676 (143904566-143995299)

Uncharacterized gene INRA0022701 144047257 0.029 Intergenic region

ASGA0029999 144089138 0.004 Intergenic region ASGA0030003 144260593 0.033 Intergenic region ALGA0037508 144303289 0.044 Intergenic region 6_144402150 144402150 0.033 Intergenic region MARC0093463 144493600 0.025 Intergenic region

37 The QTL region SSC6b appears to contain two separate clusters of significant SNPs, one between the chromosome positions 139 Mb and 141 Mb containing 12 significant SNPs;

block 1, and one centered on chromosome position 144 Mb containing 10 significant SNPs;

block 2 (Figure 11).

A

B

C

Figure 11: A. Association analysis linking 230 SNPs on porcine chromosome 6 to occurrence of hernia. A p-value of 0.05 (grey dashed line) was considered significant. The blue shadowing signifies the two blocks in the region, and the orange shadowing signifies the region with elevated read depth values; HRD-block.

B. Map of genes and their orientation in the region, based on information available from NCBI Gene. Genes containing significant SNPs are marked in red. The map is scaled to chromosome positions shown in A. C. An LD plot was constructed using Haploview. R² was used as a measurement of LD. The color gradient of the diamonds represent the R² value, with R² = 0 shown as white and R² = 1 shown as black.

38

The pattern of LD between significant SNPs is not as clear for region SSC6b as for region SSC6a. For both clusters of significant SNPs there is high LD between as many as 5 SNPs, but weak LD between those SNPs and other significant SNPs within the same block.

Within the first block of SNPs there are three significant SNPs (Haploview position 14, 40, and 41) that don’t show high LD with any other significant SNPs in the same region (Figure 12, A and B), two of which are located within a small region with generally high LD (Figure 12, B). There are five significant SNPs that show generally high LD, three of which are in complete LD with each other. Within the same region, two more significant SNPs are in high LD, but show little or no LD with the other five significant SNPs (Figure 12, C).

39 Figure 12: Sections (A, B, and C) of the LD plot constructed for region SSC6b using Haploview, displaying block 1. The sections are from the first cluster of significant SNPs, located between chromosome positions 139 Mb and 141 Mb. R² was used as a measurement of LD. The color gradient of the diamonds represent the R² value, with R² = 0 shown as white and R² = 1 shown as black. The significant SNPs within the sections are indicated with red rectangles. The intersections of the yellow or orange lines show the degree of LD between the significant SNPs, and the topmost yellow and orange lines connect SNPs in high LD.

A B

C

40

Within the second cluster of significant SNPs (block 2), five SNPs show very high LD with each other (Figure 13, complete yellow lines), and moderate LD with the SNP with the lowest p-value (Figure 13, dashed yellow line). In the same region, two more significant SNPs are in compete LD, but show little or no LD with the other significant SNPs (Figure 13, orange line). The SNP with the lowest p-value are in moderate LD with one other SNP as well (Figure 13, dashed green line).

Figure 13: Section of the LD plot constructed for region SSC6b using Haploview, displaying block 2. The section shows the second cluster of significant SNPs; centered on chromosome position 144 Mb. R² was used as a measurement of LD. The color gradient of the diamonds represent the R² value, with R² = 0 shown as white and R² = 1 shown as black. The significant SNPs within the section are indicated with red rectangles. The intersections of the yellow, orange or green lines show the degree of LD between the significant SNPs, and the topmost yellow, orange and green lines connect SNPs in high LD. The SNP with the lowest p-value from the association analysis is indicated with a filled, red rectangle, and is in moderate LD with other SNPs connected by the yellow line, as well as the SNP with Haploview position 223.

41 Table 9: Results from Tagger analysis for significant SNPs in region SSC6b. R² is

the level of LD between the significant SNP (Allele) and the tagSNP (Test).

Allele Test

SNP name Chr. position SNP name Chr. position R² MARC0037985 138852864 MARC0037985 138852864 1.0 6_138924570 138924570 6_139077739 139077739 0.816 6_139077739 139077739 6_139077739 139077739 1.0 6_139626802 139626802 6_139626802 139626802 1.0 6_139664663 139664663 6_140722873 140722873 0.813 ALGA0115609 140397044 ALGA0115609 140397044 1.0 ASGA0104579 140475536 ALGA0114933 140483422 1.0 ASGA0091723 140480675 ALGA0114933 140483422 1.0 ALGA0114933 140483422 ALGA0114933 140483422 1.0 MARC0025692 140511844 MARC0025692 140511844 1.0 ASGA0029808 140603308 MARC0025692 140511844 0.972 6_140722873 140722873 6_140722873 140722873 1.0 6_143795121 143795121 ASGA0030003 144260593 0.952 ASGA0095712 143814685 ASGA0030003 144260593 0.952 6_143889528 143889528 ASGA0030003 144260593 1.0 MARC0016562 143952619 INRA0022701 144047257 1.0 INRA0022701 144047257 INRA0022701 144047257 1.0 ASGA0029999 144089138 ASGA0029999 144089138 1.0 ASGA0030003 144260593 ASGA0030003 144260593 1.0 ALGA0037508 144303289 ALGA0037508 144303289 1.0 6_144402150 144402150 ASGA0030003 144260593 1.0 MARC0093463 144493600 MARC0093463 144493600 1.0

Table 9 shows the results from the Tagger analysis; ultimately, 12 SNPs (Table 10) are capable of capturing 100 % of the alleles of the significant SNPs in SSC6b.

Table 10: Results from Tagger analysis; the 12 significant SNPs that captured 100 % of the alleles of the significant SNPs in SSC6b.

SNP name Chr. position

42